Medium-chain acyl-CoA dehydrogenase (MCAD,EC 1.3.99.3) is a mitochondrial flavoenzyme which catalyzes the initial, rate-limiting, reaction in fatty acid beta-oxidation. Inherited MCAD deficiency, a cause of fasting coma, liver dysfunction, and sudden death in childhood, reflects the importance of this enzyme in energy metabolism. The MCAD gene is highly regulated, in parallel with fatty acid oxidation rates, among tissues and during development. Elucidation of the mechanisms involved in the regulation of the MCAD gene will provide insights relevant to MCAD deficiency and to the understanding of mechanisms involved in the regulation of expression of nuclear genes encoding mitochondrial enzymes involved in fatty acid metabolism under normal conditions and in a variety of physiologic and disease states. The major goals of this proposal include the structural and functional characterization of the promoter and upstream cis-acting regulatory regions of the human MCAD gene including identification of the elements involved in tissue-specific, developmental, and retinoic acid-responsive transcriptional regulation. Ultimately, we hope to identify regulatory sequences and transacting regulatory DNA binding proteins involved in coordinate control of genes encoding metabolic enzymes and mitochondrial proteins. The promoter region and regulatory elements will be characterized by transfecting a variety of chimeric plasmids containing varying lengths of MCAD gene 5'-flanking DNA fused to the bacterial chloramphenicol acetyltransferase (CAT) gene into mammalian cells derived from several tissues with differing MCAD mRNA expression levels. Retinoic acid response elements will be localized by identifying the regulatory sequences of the MCAD gene which confer retinoic acid-responsive transcriptional activation to the MCAD-CAT plasmids in mammalian cells in culture. The regulatory sequences will be compared to sequences of known regulatory elements in other genes, particularly those encoding enzymes involved in metabolism. The regulatory elements will be further characterized by performing DNA-nuclear protein binding assays. Ultimately, the trans-acting regulatory proteins involved in the transcriptional regulation of the MCAD gene will be identified by isolating and characterizing their cDNAs. The role of the regulatory elements in developmental and tissue-specific regulation of the MCAD gene in vivo will be evaluated by employing a transgenic mouse system.